Spinal cord injury (SCI) currently affects approximately 250,000 people in the U.S., with more than 10,000 new cases occurring each year. Currently, there is no cure for SCI. This study seeks to establish appropriate therapeutic interventions in a chronic SCI situation to support neuroanatomical and neurophysiological recovery. Work from the current funding period further defined the neuroprotective role of glial cell line-derived neurotrophic factor (GDNF) after an acute injury, the use of matrix-degrading enzymes to modify a spinal cord injury site, and the response of chronically injured neurons to neurotrophic factor (NTF) therapy after a second injury. This proposal will build upon this work and progress into new territory by addressing the significance of modulating an SCI site before applying strategies designed to accelerate axonal regrowth. We will also attempt to reduce some negative aspects of the neuronal response to injury by treating an acute injury with NTFs to heighten the regenerative effort of these neurons at chronic stages injury. The Specific Aims address the hypotheses that 1) degradation of the extracellular matrix prior to NTF treatment will increase axonal regeneration in the injured spinal cord, 2) acute neuroprotective therapy will enhance strategies for neuroregeneration in a chronic injury situation, and 3) regeneration enhanced by combined acute and chronic interventions can be characterized by changes in expression of regeneration-associated genes and/or activation of specific signaling pathways. Aim I will use an established cervical SCI-peripheral nerve (PN) graft model to test whether modulation of the extracellular matrix of the injured spinal cord prior to use of NTFs enhances axonal outgrowth from a PN graft and whether matrix-degrading enzymes effectively treat an established glial scar. Aim II examines acute neuroprotective treatment with GDNF to promote long-term survival and regeneration of chronically injured neurons. In Aim III, possible mechanisms underlying GDNF-enhanced regeneration after chronic injury will be examined by studying the expression of regeneration-associated genes and activated signaling pathways using laser microdissection, quantitative RT-PCR, and Western blot procedures. Overall, these experiments will provide fundamental information about cellular and molecular aspects of the neuronal and non-neuronal responses to long-term injury that will be instrumental in designing intervening therapies to repair the chronically injured spinal cord.